The present invention relates generally to novel protein recovery and purification methods and more specifically to novel methods for the recovery and purification of factor IX.
The advent of recombinant technology now allows for the production of high levels of proteins within suitably transformed host cells. For secreted proteins, purification of the protein of interest involves isolation and purification from the host cell culture medium. Typically, the culture medium contains selected nutrients (e.g. vitamins, amino acids, cofactors, minerals,) and additional growth factors/supplements including insulin and possibly additional exogenous proteins. Conditioned medium contains not only the secreted product of interest, but also significant quantities of additional secreted host cell proteins and other substances (e.g. nucleic acids, membrane vesicles). Although expressed at high levels, the product of interest may represent a minority of all proteins present in conditioned medium. Not unexpectedly, proteins secreted by transformed host cells may possess characteristics quite different from those of the product of interest (e.g. charge, molecular size, amino acid composition). Similarly, selected secreted host cell proteins may exhibit properties very similar to those of the product of interest, thereby placing significant burden on the process used for purification. While developing a process for purification of a recombinant protein from conditioned medium, it is important that conditions used be limited with respect to denaturation of the product of interest (conditions which could be used to exploit minor differences between secreted proteins for major benefit to separation), thereby making it difficult to separate the product of interest from all other host cell proteins present.
In addition to secreted host cell proteins described above, conditioned medium may also contain products derived from the heterologously-expressed gene coding for the product of interest. These are not desirable for the final drug substance and include, for example, product forms lacking certain post-translational modifications such as glycosylation, sulfation, gamma carboxylation, or other modification potentially necessary for biological activity. In addition, proteolytically-degraded forms of the product of interest may be present in conditioned medium which also need to be removed during purification, but which very closely resemble the product of interest. Unfortunately, most approaches, such as ion exchange chromatography, hydrophobic interaction chromatography, and size exclusion chromatography may not provide the extent of resolution of the product of interest necessary for use in human therapeutic situations from these undesired forms. To take full advantage of minor differences between the desired product and contaminants (e.g. small charge differences, small differences in molecular size) the use of strong denaturants is often required. Such denaturants, however, can lead to loss of biological activity, expression of neoantigenic sites, and potentially enhance chemical decomposition of selected post-translational modifications.
In addition to separating the product of interest from molecules with similar properties (e.g. modified forms of the expressed gene), it is also important to recognize the need to separate the desired product from components present in conditioned medium with which it specifically interacts. Where the protein of interest is positively charged, it will tend to bind to any negatively charged molecules present thereby making purification of the protein by traditional methods very difficult.
Of general background interest to the present invention are the following. Yan, U.S. Pat. No. 4,981,952 (Jan. 1, 1991) and Yan, etal. Bio/Technology 8:655 (July 1990) which disclosed the use of pseudo-affinity anion exchange chromatography for the purification of vitamin K-dependent proteins. Josic, et al. J. Chrorn. 632:1 (1993) disclosed the use of heparin affinity chromatography to resolve factor IX from other vitamin K-dependent proteins. Suomela. Thromb. Res. 7:101 (1975); Suomela, Eur. J. Bio. Chem. 71:145 (1976); and Suomela, Thrombos. Haemostis. 35:211 (1976) described the use of hydroxyapatite in the separation of various clotting factors and factor IX plasma variants (based on charge differences due to variation in content of carbohydrate moieties, for example, sialic acid and galactose). However, Reekers, et al. Haemostasis 1:2 (1972) demonstrated the inability of hydroxyapatite to separate factors II, VII and IX from each other and from other plasma proteins. Schwinn, et al. U.S. Pat. No. 4,411,794 disclosed the partial purification of blood clotting factors using hydroxyapatite in the presence of calcium at a concentration of 50-200 mM. Feldman, et al. Biotech. Blood Proteins 227:63 (1993) and Roberts, et al. Vox Sang 67(suppl. 1): 69 (1994) disclosed the reduction of viral infectivity using acidification and copper-charged chelating Sepharose which resulted in low factor IX yields from human plasma.
Typically, researchers have used combinations of traditional chromatographic techniques to purify desired products. Often times, such techniques are not sufficient for purification of a product to the level of purity and consistency desired for a human therapeutic product. Researchers have attempted to overcome this difficulty by use of affinity chromatography wherein a protein of interest is bound to an immobilized ligand with which it interacts specifically. Following appropriate washing, the desired product can be eluted by disruption of the ligand-protein interaction, often resulting in a significantly more pure eluate. However, in the instance of separation of a desired product from modified forms present in conditioned medium, single step affinity chromatographic techniques may not be sufficient, and must be used in conjunction with other affinity resins and/or traditional separation techniques. Even high resolution affinity chromatography steps (e.g., immunoaffinity purification using an immobilized monoclonal antibody) may not afford sufficient resolution of the desired product from other components due to common sites of interaction (e.g., where an epitope which is present in the product of interest, is present as well in a proteolytically-degraded form of the product).
Accordingly, there continues to exist a need in the art for protein purification methods that effectively overcome such difficulties.
Provided by the present invention are methods for the purification of factor IX in a solution comprising the steps of applying the solution containing factor IX to an anion exchange resin, washing said anion exchange resin with a solution having a conductivity that is less than required to elute factor IX from the resin, eluting said anion exchange resin with a first eluant to form a first eluate, applying said eluate to a heparin or heparin-like (e.g., negatively charged matrix) resin, eluting said heparin or heparin-like resin with a second eluant to form a second eluate, applying said second eluate to an hydroxyapatite resin, and then eluting said hydroxyapatite resin with a third eluant to form a third eluate containing the purified factor IX. Optionally, the first eluate can be applied to an hydroxyapatite resin. As yet another option, the method comprises the further steps of applying the third eluate to an immobilized metal affinity resin, and then eluting the immobilized metal affinity resin with a fourth eluant to form a fourth eluate containing the purified factor IX. According to the methods of the invention, the factor IX can be either plasma-derived, expressed by cells in culture, or recombinantly produced as is known to one skilled in the art. Preferably, the first wash comprises a solution having a conductivity that is less than required to elute factor IX from the column and is generally greater than or equal to the conductivity of the load solution and of the first eluant buffer; this conductivity is sufficient to remove a substantial proportion of those contaminating proteins that would otherwise be present in the first eluate. A suitable first wash comprises a salt solution such as sodium chloride, potassium chloride, sodium sulphate, sodium phosphate, or potassium phosphate, and optionally, may contain a suitable buffering agent. Suitable concentration ranges are those which are effective in removing contamininants without eluting factor IX and include for example 25 mM to 200 mM salt, and preferably is 200 mM sodium chloride. The first eluant comprises a divalent cation such as calcium, magnesium, manganese, strontium, zinc, cobalt, and nickel; suitable concentration ranges are those which are effective in eluting factor IX, including for example a solution containing a buffering agent at pH about 8.0 such as Tris, in the range of 5 to 100 mM, preferably approximately 50 mM, a salt such as NaCl in the range of 50 to 250 mM, preferably 100 mM, and calcium chloride in the range of 5 to 20 mM, preferably approximately 10 mM.
Suitable anion exchange resins include those resins having a positively charged group such as diethyleaminoethane (DEAE), polyethyleneimine (PEI), and quaternary aminoethane (QAE) and include Q-Sepharose Fast Flow, DEAE-Sepharose Fast Flow, POROS-Q, Fractogel-TMAE, Fractogel-DMAE, and QAE-Toyopearl, with the preferred resin being Q-Sepharose Fast Flow (Pharmacia).
The second eluant can be a suitable salt in buffer, such as Tris with sodium chloride and potassium chloride, with 50 mM TRIS, 0.50 M NaCl, pH 8.0 being preferred. Suitable heparin or heparin-like resins include those resins having a negatively charged group such as heparin, sulfated esters of cellulose, sullfylpropyl (SP), carboxyl, and carboxy methyl and include Matrex Cellufine Sulfite, Heparin Sepharose, Heparin Toyopearl, Carboxy Sulfon, Fractogel EMD-SO3, and Fractogel-EMD COO, with the preferred being Matrex Cellufine Sulfate.
The third eluant can be a salt, such as phosphate and sulphate, with 0.5 M potassium phosphate, 0.2 M NaCl, pH 7.2 preferred. Suitable hydroxyapatite resins include any containing calcium-phosphate such as ceramic-Hy(droxyapatite, Biogel HT, and others, with ceramic-HA preferred. The immobilized metal affinity resin can be one such as Fractogel-EMD-Chelate, Chelating-Sepharose, Matrex Cellufine Chelate, and POROS 20 MC, with Fractogel EMD-Chelate currently preferred. The fourth eluant is a buffer solution containing a chelator such as imidazole, EDTA, EGTA, glycine, histidine, and Tris, with the preferred being 20 mM potassium phosphate, 15 mM imidazole, 0.1 M NaCl, pH 7.1.
Also provided by the present invention are factor IX compositions produced by the methods of the invention. The factor IX so produced has a specific activity in the range of 240-400 U/mg, and is optionally about 240 U/mg.
As used herein, the term xe2x80x9cfactor IXxe2x80x9d includes, but is not limited to factor IX isolated from plasma, transformed cell lines, and recombinantly produced factor IX isolated from host cell culture medium.
As used herein, the term xe2x80x9canion exchange resinxe2x80x9d includes, but is not limited to resins having a positively charged moiety (at neutral pH), such as diethyleaminoethane (DEAE), polyethyleneimine (PEI), and quaternary aminoethane (QAE) and includes, for example, Q-Sepharose Fast Flow (Pharmacia), DEAE-Sepharose Fast Flow, DEAE-Toyopearl, QAE-Toyopearl, POROS-Q, Fractogel-DMAE, Fractogel EMD-TMAE, Matrex Cellufine DEAE and the like.
As used herein, the term xe2x80x9cfirst washxe2x80x9d includes, but is not limited to a solution having a conductivity that is less than required to elute factor IX from the anion exchange column and whose conductivity is generally greater than or equal to the conductivity of the load solution and of the conductivity of the first eluant; this conductivity is sufficient to remove a substantial proportion of those contaminating proteins that would otherwise be present in the eluate. As one skilled in the art readily appreciates, the first wash can be any salt solution and includes, for example, sodium chloride, potassium chloride, sodium sulphate, sodium phosphate, or potassium phosphate, and can be suitably buffered. Typically, concentrations range from low (25 mM salt) to high (200 mM salt), with 200 mM sodium chloride presently preferred.
As used herein, the term xe2x80x9cfirst eluantxe2x80x9d includes, but is not limited to solutions composed of a buffering agent (e.g. Tris) at a concentration of approximately 0.05 M, salt (e.g. NaCl) at a concentration which is not sufficient for elution from the resin in the absence of divalent cation (e.g. approximately 0.10 M-0.20 M), and divalent cation (e.g. CaCl2) at low concentrations of approximately 0.01 M, at pH 8.0. The selection of buffer composition is compatible with the presence of divalent cation. Preferably, the xe2x80x9cfirst eluantxe2x80x9d has a lower conductivity than the xe2x80x9cfirst washxe2x80x9d.
As used herein, the terms xe2x80x9cheparinxe2x80x9d resin and xe2x80x9cheparin-likexe2x80x9d resin are used interchangeably, and include but are not limited to, resins containing an immobilized negatively charged moiety such as heparin, sulfated esters of cellulose, sulfylpropyl (SP), carboxyl, and carboxy methyl and includes Fractogel-EMD-SO3, Carboxy Sulfon, Fractogel-EMD-COO, Heparin-Sepharose, and Matrex Cellutfine Sulfate.
As used herein, the term xe2x80x9csecond eluantxe2x80x9d includes, but is not limited to: solutions composed of a buffering agent (e.g. Tris) at a concentration of approximately 0.05 M, and salt (e.g. NaCl, KCl, Na2SO4) at a concentration sufficient to disrupt the interaction of factor IX with the negatively-charged resin support (e.g. 0.50 M) at approximately pH 8.0. As used in this process, the second eluant should be compatible with the subsequent process step, i.e., hydroxyapatite.
As used herein, the term xe2x80x9chydroxyapatite columnxe2x80x9d includes, but is not limited to: calcium phosphate gel supports including for example, BioGel-HT, and Ceramic-hydroxyapatite.
As used herein, the term xe2x80x9cthird eluantxe2x80x9d (and xe2x80x9csecond phosphate bufferxe2x80x9d) includes, but is not limited to: solutions composed of a buffering agent (e.g. phosphate or sulfate) at concentrations sufficient to disrupt interaction of factor IX with the resin (e.g. approximately 0.20 M or higher) and salt (e.g. NaCl, KCl) present at concentrations sufficient to minimize charge-interactions of the factor IX with the hydroxyapatite resin, at approximately neutral pH (pH 7.2); the term xe2x80x9cfirst phosphate bufferxe2x80x9d includes but is not limited to solutions composed of a buffering agent (e.g., phosphate or sulfate) at concentrations sufficient to remove inactive forms of factor IX from the hydroxy-apatite resin.
As used herein, the term xe2x80x9cimmobilized metal affinity resinxe2x80x9d (IMAC) includes, but is not limited to: resins containing an immobilized functional moiety (e.g. iminodiacetic acid) capable of binding and coordinating multivalent cations including Chelating-Sepharose, Fractogel-EMD-Chelate, POROS 20 MC, and Matrex Cellufine Chelate. The bound metal ion can be selected from several possible choices including but not limited to copper, nickel, cadmium, cobalt, iron, zinc, or strontium.
The term xe2x80x9cfourth eluantxe2x80x9d (also termed xe2x80x9cdisplacerxe2x80x9d) includes but is not limited to, any compound which will displace bound factor IX from the IMAC resin support, while minimizing displacement of the immobilized metal ion from the resin support, and includes but is not limited to such compounds as glycine, histidine, tris, imidazole, EDTA, EGTA, and the like. As one skilled in the art readily appreciates, the appropriate concentration of displacer will vary according to binding affinity and can be ascertained by experimental evaluation of conditions. Typically, concentrations range from low (e.g. 5-15 mM displacer) to high (e.g. 100-200 mM displacer).
Reference to factor IX specific activity of xe2x80x9cU/mgxe2x80x9d includes but is not limited to: biological activity determined in the in vitro (APTT) clotting assay using pooled plasma or isolated, purified factor IX as standard. The concentration of protein can be determined by any of several appropriately validated methods including SEC, RP-HPLC, dye-based assays (e.g., Bradford, Lowry) or absorbance at 280 nm. Factor IX activity is determined according to the method of Pittman, D., et al., Blood 79:389-397 (1992) utilizing factor IX-deficient plasma.
FIG. 1 provides an overview of the process. While the order of the steps set forth is the presently preferred embodiment, it will be appreciated by one skilled in the art that the order can be re-configured if desired and that steps can be omitted.
According to the present invention, cells are first removed from conditioned medium, e.g. by microfiltration (MF) utilizing tangential flow filtration membranes with pore size of approximately 0.6 xcexcm. Optionally, cell-free conditioned medium is prepared for purification by filtering through a 0.45 xcexcm depth filter. The cell-free conditioned medium can then be concentrated by ultrafiltration, if desired, followed by diafiltration into an appropriate buffer for loading onto the first chromatographic step. Alternatively, the cell-free conditioned medium may be loaded directly onto the first chromatography column equilibrated in an appropriate buffer. 
The initial process step, UF/DF#1, entails concentration of the cell-free factor IX-containing conditioned media by ultrafiltration, followed by diafiltration. Although not required for binding of factor IX to the first chromatography column, this step is effective in removing small-molecular-weight cell culture media components. Such components may bind to the initial chromatography column, thereby decreasing the capacity of the column for factor IX. UF/DF#1 is used to exchange the factor IX into an appropriate buffer solution for subsequent processing.
In the first chromatography step, anion-exchange on Q-Sepharose Fast Flow (FF) (Pharmacia), the factor IX is captured and purified from host-cell components present in the UF/DF #1 concentrated pool. The Q-Sepharose FF column adsorbs the factor IX protein, and contaminating host-cell proteins with isoelectric points greater than the operating pH are removed from the process stream by flowing through the column. The column to which factor IX is adsorbed is then washed prior to elution to remove loosely-bound contaminants and adjust the conductivity of the buffer in preparation for elution.
Typically, bound proteins are eluted from Q-Sepharose FF by increasing the ionic strength of the buffer. The factor IX purification process, however, employs this resin in a pseudo-affinity anion-exchange mode in which active factor IX is eluted by addition of e.g., calcium chloride to the buffer. This divalent cation results in elution of active forms of factor IX from the resin. Some less active forms of factor IX may also elute from the Q-Sepharose FF column with this elution buffer. Selected inactive forms of factor IX and other, contaminating host-cell proteins remain bound to the column. The Q-Sepharose FF step achieves a significant increase in the purity of the factor IX.
In the second chromatography step, the Q-Sepharose FF elution pool is loaded directly, without dilution, onto the Matrex Cellufine Sulfate column. The factor IX adsorbs to the column, while other, contaminating proteins (e.g., soluble PACE and other host-cell proteins present in the Q-Sepharose FF eluate) are removed from the process stream by flowing through the column. The column is washed with a low-ionic-strength buffer to remove all non-binding proteins. The factor IX is eloted by an increase in the ionic strength of the buffer, using salt (e.g., sodium chloride).
Further removal of inactive factor IX forms is obtained during the third chromatography step, Ceramic-HA column chromatography. The pH of the Matrex Cellufine Sulfate elution pool is adjusted to approximately 7.5, and the elution pool is then loaded directly onto the Ceramic-HA column. The factor IX is adsorbed by the column. The Ceramic-HA column is washed with buffer to remove loosely bound contaminants, followed by a wash with 50 mM potassium phosphate, 0.185 M NaCl (pH 7.2) to remove more tightly bound contaminants, including inactive forms of factor IX. Bound, active factor IX is eluted in a step-wise manner using a solution containing a higher concentration of potassium phosphate (e.g. 200 mM or greater, pH 7.2) as the eluant.
The fourth chromatography step, Fractogel EMD-Chelate -Cu(II) chromatography, removes low levels of contaminating host-cell proteins still present in the product stream. The Ceramic-HA elution pool is loaded directly onto the Fractogel EMD-Chelate -Cu(II) column. Factor IX and a number of contaminating, proteins are adsorbed to the column. Purified active factor IX is eluted from the column by low concentrations of imidazole (e.g. approximately 15 mM) in the buffer, and the residual, contaminating host-cell proteins are removed from the product stream by remaining bound to the column.
Finally, the Fractogel EMD-Chelate -Cu(II) elution pool is concentrated by ultrafiltration, followed by diafiltration (UF/DF#2) into a buffer identical to a formulation buffer except that it does not contain polysorbate 80. A suitable formulation buffer comprises histidine, glycine, sucrose, and polysorbate-80 optionally at 10 mM, 260 mM, 1%, and 0.005%, respectively. Upon completion of the diatfiltratiun, factor IX is concentrated to achieve a target concentration. The product pool is removed from the UF/DF 2, apparatus and formulated by addition of polysorbate 80 to a target concentration of 0.005%. The factor IX drug substance is then filtered (0.2 xcexcm), sampled, labeled. and stored frozen at approximately xe2x88x9280xc2x0 C. The last process step, UF/DF#2, is effective in concentrating and diafiltering the purified factor IX drug substance without significant protein denaturation or loss. SDS-PAGE analysis (reduced and nonreduced) is one method used to evaluate overall process performance. Each step provides greater than 80% to 100% yield and the average overall yield of factor IX is about 51%. The overall process yield is tleterminedl from the clotting activity entering the purification process and the total clotting activity in the factor IX drug substance (excluding material removed as in-process samples and retains).